An enhanced method for nucleic acid detection with CRISPR-Cas12a using phosphorothioate modified primers and optimized gold-nanopaticle strip
CRISPR-Cas12a system has been shown promising for nucleic acid diagnostics due to its rapid, portable and accurate features. However, cleavage of the amplicons and primers by the cis- and trans-activity of Cas12a hinders the attempts to integrate the amplification and detection into a single reactio...
| Main Authors: | , , , , , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
KeAi Communications Co., Ltd.
2021-12-01
|
| Series: | Bioactive Materials |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2452199X21002255 |
| _version_ | 1827279277841711104 |
|---|---|
| author | Jiaojiao Gong Lijuan Kan Xiuming Zhang Ying He Jiaqiang Pan Liping Zhao Qianyun Li Menghao Liu Jie Tian Sili Lin Zhouyu Lu Liang Xue Chaojun Wang Guanghui Tang |
| author_facet | Jiaojiao Gong Lijuan Kan Xiuming Zhang Ying He Jiaqiang Pan Liping Zhao Qianyun Li Menghao Liu Jie Tian Sili Lin Zhouyu Lu Liang Xue Chaojun Wang Guanghui Tang |
| author_sort | Jiaojiao Gong |
| collection | DOAJ |
| description | CRISPR-Cas12a system has been shown promising for nucleic acid diagnostics due to its rapid, portable and accurate features. However, cleavage of the amplicons and primers by the cis- and trans-activity of Cas12a hinders the attempts to integrate the amplification and detection into a single reaction.Through phosphorothioate modification of primers, we realized onepot detection with high sensitivity using plasmids of SARS-CoV-2, HPV16 and HPV18. We also identified the activated Cas12a has a much higher affinity to C nucleotide-rich reporter than others. By applying such reporters, the reaction time required for a lateral-flow readout was significantly reduced. Furthermore, to improve the specificity of the strip-based assay, we created a novel reporter and, when combined with a customized gold-nanopaticle strip, the readout was greatly enhanced owing to the elimination of the nonspecific signal. This established system, termed Targeting DNA by Cas12a-based Eye Sight Testing in an Onepot Reaction (TESTOR), was validated using clinical cervical scrape samples for human papillomaviruses (HPVs) detection.Our system represents a general approach to integrating the nucleic acid amplification and detection into a single reaction in CRISPR-Cas systems, highlighting its potential as a rapid, portable and accurate detection platform of nucleic acids. |
| first_indexed | 2024-04-24T08:17:23Z |
| format | Article |
| id | doaj.art-a3b5af1851194ba1a796941837a1d984 |
| institution | Directory Open Access Journal |
| issn | 2452-199X |
| language | English |
| last_indexed | 2024-04-24T08:17:23Z |
| publishDate | 2021-12-01 |
| publisher | KeAi Communications Co., Ltd. |
| record_format | Article |
| series | Bioactive Materials |
| spelling | doaj.art-a3b5af1851194ba1a796941837a1d9842024-04-17T03:20:56ZengKeAi Communications Co., Ltd.Bioactive Materials2452-199X2021-12-0161245804590An enhanced method for nucleic acid detection with CRISPR-Cas12a using phosphorothioate modified primers and optimized gold-nanopaticle stripJiaojiao Gong0Lijuan Kan1Xiuming Zhang2Ying He3Jiaqiang Pan4Liping Zhao5Qianyun Li6Menghao Liu7Jie Tian8Sili Lin9Zhouyu Lu10Liang Xue11Chaojun Wang12Guanghui Tang13Yaneng Biotech, Co., Ltd, Fosun Pharma, Shenzhen 518100, ChinaDepartment of Laboratory Medicine, Luohu District People's Hospital, Shenzhen 518001, ChinaDepartment of Laboratory Medicine, Luohu District People's Hospital, Shenzhen 518001, ChinaDepartment of Laboratory Medicine, The Eighth Affiliated Hospital, Sun Yat-sen University, Shenzhen 518033, ChinaYaneng Biotech, Co., Ltd, Fosun Pharma, Shenzhen 518100, ChinaDepartment of Laboratory Medicine, Nanning First People's Hospital, Nanning 530022, ChinaDepartment of Neurology, Hwa Mei Hospital, University of Chinese Academy of Sciences, Ningbo 315010, ChinaNanobiological Medicine Center, Key Lab of Fuel Cell Technology of Guangdong Province, School of Chemistry and Chemical Engineering, South China University of Technology, Guangzhou 510641, ChinaYaneng Biotech, Co., Ltd, Fosun Pharma, Shenzhen 518100, ChinaYaneng Biotech, Co., Ltd, Fosun Pharma, Shenzhen 518100, ChinaYaneng Biotech, Co., Ltd, Fosun Pharma, Shenzhen 518100, ChinaYaneng Biotech, Co., Ltd, Fosun Pharma, Shenzhen 518100, China; Corresponding author.Department of Urology, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China; Corresponding author.Yaneng Biotech, Co., Ltd, Fosun Pharma, Shenzhen 518100, China; Corresponding author.CRISPR-Cas12a system has been shown promising for nucleic acid diagnostics due to its rapid, portable and accurate features. However, cleavage of the amplicons and primers by the cis- and trans-activity of Cas12a hinders the attempts to integrate the amplification and detection into a single reaction.Through phosphorothioate modification of primers, we realized onepot detection with high sensitivity using plasmids of SARS-CoV-2, HPV16 and HPV18. We also identified the activated Cas12a has a much higher affinity to C nucleotide-rich reporter than others. By applying such reporters, the reaction time required for a lateral-flow readout was significantly reduced. Furthermore, to improve the specificity of the strip-based assay, we created a novel reporter and, when combined with a customized gold-nanopaticle strip, the readout was greatly enhanced owing to the elimination of the nonspecific signal. This established system, termed Targeting DNA by Cas12a-based Eye Sight Testing in an Onepot Reaction (TESTOR), was validated using clinical cervical scrape samples for human papillomaviruses (HPVs) detection.Our system represents a general approach to integrating the nucleic acid amplification and detection into a single reaction in CRISPR-Cas systems, highlighting its potential as a rapid, portable and accurate detection platform of nucleic acids.http://www.sciencedirect.com/science/article/pii/S2452199X21002255CRISPR-Cas12aOnepotVisual detection |
| spellingShingle | Jiaojiao Gong Lijuan Kan Xiuming Zhang Ying He Jiaqiang Pan Liping Zhao Qianyun Li Menghao Liu Jie Tian Sili Lin Zhouyu Lu Liang Xue Chaojun Wang Guanghui Tang An enhanced method for nucleic acid detection with CRISPR-Cas12a using phosphorothioate modified primers and optimized gold-nanopaticle strip Bioactive Materials CRISPR-Cas12a Onepot Visual detection |
| title | An enhanced method for nucleic acid detection with CRISPR-Cas12a using phosphorothioate modified primers and optimized gold-nanopaticle strip |
| title_full | An enhanced method for nucleic acid detection with CRISPR-Cas12a using phosphorothioate modified primers and optimized gold-nanopaticle strip |
| title_fullStr | An enhanced method for nucleic acid detection with CRISPR-Cas12a using phosphorothioate modified primers and optimized gold-nanopaticle strip |
| title_full_unstemmed | An enhanced method for nucleic acid detection with CRISPR-Cas12a using phosphorothioate modified primers and optimized gold-nanopaticle strip |
| title_short | An enhanced method for nucleic acid detection with CRISPR-Cas12a using phosphorothioate modified primers and optimized gold-nanopaticle strip |
| title_sort | enhanced method for nucleic acid detection with crispr cas12a using phosphorothioate modified primers and optimized gold nanopaticle strip |
| topic | CRISPR-Cas12a Onepot Visual detection |
| url | http://www.sciencedirect.com/science/article/pii/S2452199X21002255 |
| work_keys_str_mv | AT jiaojiaogong anenhancedmethodfornucleicaciddetectionwithcrisprcas12ausingphosphorothioatemodifiedprimersandoptimizedgoldnanopaticlestrip AT lijuankan anenhancedmethodfornucleicaciddetectionwithcrisprcas12ausingphosphorothioatemodifiedprimersandoptimizedgoldnanopaticlestrip AT xiumingzhang anenhancedmethodfornucleicaciddetectionwithcrisprcas12ausingphosphorothioatemodifiedprimersandoptimizedgoldnanopaticlestrip AT yinghe anenhancedmethodfornucleicaciddetectionwithcrisprcas12ausingphosphorothioatemodifiedprimersandoptimizedgoldnanopaticlestrip AT jiaqiangpan anenhancedmethodfornucleicaciddetectionwithcrisprcas12ausingphosphorothioatemodifiedprimersandoptimizedgoldnanopaticlestrip AT lipingzhao anenhancedmethodfornucleicaciddetectionwithcrisprcas12ausingphosphorothioatemodifiedprimersandoptimizedgoldnanopaticlestrip AT qianyunli anenhancedmethodfornucleicaciddetectionwithcrisprcas12ausingphosphorothioatemodifiedprimersandoptimizedgoldnanopaticlestrip AT menghaoliu anenhancedmethodfornucleicaciddetectionwithcrisprcas12ausingphosphorothioatemodifiedprimersandoptimizedgoldnanopaticlestrip AT jietian anenhancedmethodfornucleicaciddetectionwithcrisprcas12ausingphosphorothioatemodifiedprimersandoptimizedgoldnanopaticlestrip AT sililin anenhancedmethodfornucleicaciddetectionwithcrisprcas12ausingphosphorothioatemodifiedprimersandoptimizedgoldnanopaticlestrip AT zhouyulu anenhancedmethodfornucleicaciddetectionwithcrisprcas12ausingphosphorothioatemodifiedprimersandoptimizedgoldnanopaticlestrip AT liangxue anenhancedmethodfornucleicaciddetectionwithcrisprcas12ausingphosphorothioatemodifiedprimersandoptimizedgoldnanopaticlestrip AT chaojunwang anenhancedmethodfornucleicaciddetectionwithcrisprcas12ausingphosphorothioatemodifiedprimersandoptimizedgoldnanopaticlestrip AT guanghuitang anenhancedmethodfornucleicaciddetectionwithcrisprcas12ausingphosphorothioatemodifiedprimersandoptimizedgoldnanopaticlestrip AT jiaojiaogong enhancedmethodfornucleicaciddetectionwithcrisprcas12ausingphosphorothioatemodifiedprimersandoptimizedgoldnanopaticlestrip AT lijuankan enhancedmethodfornucleicaciddetectionwithcrisprcas12ausingphosphorothioatemodifiedprimersandoptimizedgoldnanopaticlestrip AT xiumingzhang enhancedmethodfornucleicaciddetectionwithcrisprcas12ausingphosphorothioatemodifiedprimersandoptimizedgoldnanopaticlestrip AT yinghe enhancedmethodfornucleicaciddetectionwithcrisprcas12ausingphosphorothioatemodifiedprimersandoptimizedgoldnanopaticlestrip AT jiaqiangpan enhancedmethodfornucleicaciddetectionwithcrisprcas12ausingphosphorothioatemodifiedprimersandoptimizedgoldnanopaticlestrip AT lipingzhao enhancedmethodfornucleicaciddetectionwithcrisprcas12ausingphosphorothioatemodifiedprimersandoptimizedgoldnanopaticlestrip AT qianyunli enhancedmethodfornucleicaciddetectionwithcrisprcas12ausingphosphorothioatemodifiedprimersandoptimizedgoldnanopaticlestrip AT menghaoliu enhancedmethodfornucleicaciddetectionwithcrisprcas12ausingphosphorothioatemodifiedprimersandoptimizedgoldnanopaticlestrip AT jietian enhancedmethodfornucleicaciddetectionwithcrisprcas12ausingphosphorothioatemodifiedprimersandoptimizedgoldnanopaticlestrip AT sililin enhancedmethodfornucleicaciddetectionwithcrisprcas12ausingphosphorothioatemodifiedprimersandoptimizedgoldnanopaticlestrip AT zhouyulu enhancedmethodfornucleicaciddetectionwithcrisprcas12ausingphosphorothioatemodifiedprimersandoptimizedgoldnanopaticlestrip AT liangxue enhancedmethodfornucleicaciddetectionwithcrisprcas12ausingphosphorothioatemodifiedprimersandoptimizedgoldnanopaticlestrip AT chaojunwang enhancedmethodfornucleicaciddetectionwithcrisprcas12ausingphosphorothioatemodifiedprimersandoptimizedgoldnanopaticlestrip AT guanghuitang enhancedmethodfornucleicaciddetectionwithcrisprcas12ausingphosphorothioatemodifiedprimersandoptimizedgoldnanopaticlestrip |